Note: Descriptions are shown in the official language in which they were submitted.
CA 02669913 2011-09-15
ENDOXIFEN METHODS AND COMPOSITIONS
FIELD OF THE INVENTION
[0001] The invention relates to the use of endoxifen in the treatment of
mammalian
diseases. The invention also relates to liposomes and other formulations such
as complexes,
vesicles, emulsions, micelles and mixed micelles of endoxifen, methods of
preparation, and
uses, e.g., in the treatment of human and animal breast diseases. The
invention in particular
relates to compositions comprising endoxifen-lipid complexes, methods of
preparation, and
their use for the treatment of breast diseases, in particular benign and
malignant breast
disease, enhancing disease regression and reducing risk of patients developing
breast
cancer. The invention further relates to methods of preparing endoxifen and
use of
endoxifen prepared by inventive method in the treatment of human and animal
diseases.
BACKGROUND OF THE INVENTION
[0002] Every year more than 210, 000 women in the United States develop breast
cancer.
One in eight women in the US will develop breast cancer during their lives.
Approximately
70 percent of breast cancers are fueled by estrogen, and many are treated with
Tamoxifen, a
drug designed to block the effects of estrogen in breast tissue.
[0003] Tamoxifen is an anti-estrogenic drug prescribed for long-term, low dose
therapy of
breast cancer. It has been widely used for more than 30 years for the
endocrine treatment of
all stages of hormone receptor-positive breast cancer (1-2). Tamoxifen has
also been
approved for the prevention of breast cancer (3). In women, one of the adverse
events
associated with Tamoxifen is hot flashes. The risk of hot flashes is two to
three-folds
higher among women who take Tamoxifen than it is for those who do not (4).
Selective
serotonin-reuptake inhibitor (SSRI) antidepressants are prescribed to treat
hot flashes.
However, some SSRIs, such as paroxetine and fluoxetine, are known to inhibit
cytochrome
P450 (CYP) 2D6 (5), an enzyme that is important for the metabolism of many
drugs,
including Tamoxifen. Our understanding of Tamoxifen metabolism and effect has
changed
clinical practice through the wide spread recognition that the co-prescription
of drugs that
inhibit CYP2D6 may compromise Tamoxifen efficacy. It is known that co-
administration
1
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
of paroxetine decreases the plasma concentration of an active metabolite of
Tamoxifen, 4-
hydroxy-N-desmethyl-Tamoxifen (endoxifen).
[0004] Endoxifen is generated via CYP3A4-mediated N-demethylation and CYP2D6
mediated hydroxylation of Tamoxifen (see, e.g., Figure 3). Any drug that can
be substrate
of CYP3A4 or CYP2D6, especially CYP2D6 e.g., SSRIs, can decrease the level of
endoxifen (6) and thus reduce the therapeutic benefits of Tamoxifen.
Therefore, to avoid
such drug-drug interactions, one should not give them together.
[0005] Recently, endoxifen has been shown to be anti-estrogenic in breast
cancer cells
and to be more potent than Tamoxifen (7). In patients treated with Tamoxifen,
endoxifen is
present in higher concentration (12.4 ng/mL) than 4-OH-tamoxifen (1 ng/mL) in
the human
plasma. The majority of genes affected by endoxifen are estrogen-regulated
genes (8 -9).
Use of endoxifen e.g., in place of Tamoxifen, avoids several metabolic steps
that rely on
CYP2D6.
[0006] A strong need exists for methods to treat and to prevent breast
diseases without
significant adverse systemic side effects, particularly in the premenoposal
population. In
particular, there is a need for breast cancer treatments and preventatives
that having reduced
interactive effect with other medications.
SUMMARY OF THE INVENTION
[0007] The present invention provides methods and compositions for the
syntheses and
use of active agents such as anticancer agents. The present invention relates
to methods and
compositions related to the formulations and uses of endoxifen, particularly
in applications
related to the treatment or prevention of cancer.
[0008] In some embodiments, the present invention provides a method of
treating a
disease, comprising, preparing a composition comprising a therapeutically
active amount of
endoxifen and administering the composition. In some embodiments, the
endoxifen is a
free base, or is in the form of a salt. In some preferred embodiments, the
endoxifen is in the
form of a salt selected from the group of salts consisting of citrate,
acetate, formate, oxalate,
tartarate, trifluoroacetane, methane sulfonate, phosphate, sulfate, chloride,
bromide, iodide,
lactate, and formate. In some some embodiments, the endoxifen is predominantly
in a form
selected from the group consisting of E-isomer, Z-isomer, and a mixture of E-
and Z-isomer.
[0009] In some embodiments, method comprises preparing a complex comprising an
anticancer drug and at least one lipid. In some embodiments, the anticancer
drug is
2
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
endoxifen. In some embodiments, the compounds of the invention are not
complexed with
a lipid. In some embodiments, the compound is in the form of a free base or is
in the form
of a salt.
[0010] In some embodiments, the present invention provides methods of
preparing
endoxifen, comprising reacting a compound of Formula 5 with acid, wherein the
compound
of formula 5 has the structure:
CI
"I
O O
5
[0011] and, after the reaction of the compound of Formula 5 with acid,
reacting the
compound with methylamine. In some embodiments, the compound of Formula 5 is
prepared by reacting compound of formula 4
CI
O
O
4
with a compound of Formula 3.
Br
O O
3
3
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
[0012] In some embodiments, the compound of Formula 3 is prepared by reacting
compound of Formula 1
Br
OH
1
with a compound of Formula 2.
Oo
2
[0013] In some embodiments, the present invention provides methods of
purifying the
endoxifen as described above, comprising crystallizing the endoxifen and/or
chromatographically treating said endoxifen to produce a purified preparation
of endoxifen,
wherein the purified preparation of endoxifen contains predominantly E-isomer,
predominantly Z-isomer, or mixture of E- and Z-isomers of endoxifen.
[0014] As described above, in some embodiments, the invention provides
endoxfen
preparations comprising at least one lipid. In preferred embodiments, the at
least one lipid
is selected from the group consisting of egg phosphatidylcholine (EPC), egg
phosphatidylglycerol (EPG), soy phosphatidylcholine (SPC), hydrogenated soy
phosphatidylcholine (HSPC), dimyristoylphosphatidylcholine (DMPC),
dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosohatidylcholine (DPPC),
disteroylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylglycerol (DMPG),
cholesterol (Chol), cholesterol sulfate and its salts (CS), cholesterol
hemisuccinate and its
salts (Chems), cholesterol phosphate and its salts (CP),
cholesterylphosphocholine and other
hydroxycholesterol or amino cholesterol derivatives, cholesterol succinate,
cholesterol
oleate, polyethylene glycol derivatives of cholesterol (cholesterol-PEG),
coprostanol,
cholestanol, cholestane, cholic acid, cortisol, corticosterone,
hydrocortisone, and calciferol,
E-guggulsterone, Z-guggulsterone, mixture of E-and Z-guggulsterone,
monoglycerides,
diglycerides, triglycerides, carbohydrate-based lipids selected from a group
consisting of
galactolipid, mannolipid, galactolecithin, 0-sitosterol, stigmasterol,
stigmastanol, lanosterol,
a-spinasterol, lathosterol, campesterol, phosphatidylcholine,
phosphatidylglycerol,
phosphatidylethanolamine, phosphatidylserine, phosphatdylinositol,
phosphatidic acid, and
4
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
pegylated derivatives of distearoylphosphatidylglycerol,
dipalmitoylphosphatidylglycerol,
dimyristoylphosphatidylglycerol, and dioleoylphosphatidylglycerol.
[0015] In some embodiments, a composition according to the present invention
comprises endoxifen, cholesterol and/or cholesterol derivatives, and one or
more
phospholipids. In some preferred embodiments, the composition comprises a
cholesterol
derivative, and the cholesterol derivative is cholesteryl sulfate. In some
embodiments, at
least one of the phospholipids is hydrogenated soy phosphatidylcholine or soy
phosphatidylcholine.
[0016] In some embodiments of the methods and compositions of the present
invention,
the composition comprises a form selected from the group consisting of powder,
solution,
emulsion, micelle, liposome, lipidic particle, gel, and paste form. In some
preferred
embodiments, the preparing of the composition comprising a complex comprises
preparing
said complex in a lyophilized form. In some embodiments, the preparing the
complex in a
lyophilized form comprises using a cryoprotectant, wherein said cryoprotectant
comprises
one or more sugars selected from the group consisting of trehalose, maltose,
lactose,
sucrose, glucose, and dextran. In some embodiments, the composition comprises
a tablet or
a filled capsule, wherein said tablet or filled capsule optionally comprises
an enteric coating
material.
[0017] In some embodiments of the treatment methods of the present invention,
the
disease is caused by cancer or by cancer-causing agents, while in some
embodiments, the
disease is benign breast disease.
[0018] In some embodiments, the administering comprises oral, intravenous,
subcutaneous, percutaneous, parenteral, intraperitoneal, rectal, vaginal,
and/or topical
delivery said composition to said subject.
[0019] In some embodiments, the composition comprises a penetration enhancer,
wherein said penetration enhancer comprises at least one saturated or
unsaturated fatty acid
ester.
[0020] In some embodiments, the composition comprising endoxifen is formulated
in a
hydroalcoholic gel, a hydroalcoholic solution, a patch, a cream, an emulsion,
a lotion, an
ointment, a powder or an oil.
5
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
[0021] In some embodiments, the composition comprising endoxifen is formulated
in a
hydroalcoholic composition containing a penetration enhancer, an aqueous
vehicle, an
alcoholic vehicle and a gelling agent.
[0022] In some embodiments, the hydroalcoholic composition comprises a
neutralizing
agent.
[0023] In some embodibments, the hydroalcoholic composition comprises
endoxifen at
about 0.0 1% to 0.20% by weight; isopropyl myristate at about 0.1 % to 2.0%,
preferably
0.5% to 2.0% by weight; alcohol at about 50.0% to 80.0%, preferably about
60.0% to
75.0% by weight; aqueous vehicle at about 20.0% to 60.0%, preferably 25.0% to
50.0% by
weight; and gelling agent at about 1.0% to 10.0%, preferably about 0.5% to
5.0% by
weight. In some embodiments, the wherein the percentage of components is
weight to
weight of the composition.
[0024] In some embodiments, the alcohol is ethanol or isopropanol, and
constitutes in
absolute form.
[0025] In some embodiments, the aqueous vehicle is a phosphate buffered
solution.
[0026] In some embodiments, the gelling agent is selected from the group
consisting of
polyacrylic acid, hydroxypropylcellulose and a cellulose derivative other than
hydroxypropylcellulose.
[0027] In some embodiments, the hydroalcoholic composition further comprises a
neutralizing agent, wherein said neutralizing agent is selected from the group
consisting of
sodium hydroxide, potassium hydroxide, ammonium hydroxide,
aminomethylpropanol,
arginine, trolamine, and tromethamine, and wherein said neutralizing agent
exists at a
neutralizing agent/gelling agent ratio of about 1:1 to about 4:1.
[0028] In some embodiments, the invention provides methods of delivering
endoxifen,
comprising: providing any of the above described compositions and delivering
the
composition so as to expose the composition to a cell.
[0029] In some embodiments, the cell is in vivo.
[0030] In some embodiments of the invention, the host is a mammal.
[0031] The present invention also provides methods of inhibiting hormone-
dependent
breast carcinoma in a mammal comprising administering any of the above
compositions to
the mammal.
[0032] The present invention further provides methods of inhibiting a cancer
in a
mammal, said cancer including, but not limited to, lung cancer, colon cancer,
breast cancer,
leukemia, renal cancer, melanoma, cancer or the central nervous system, and
prostate cancer
6
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
in a mammal; the method comprising administering any of the above compositions
to said
mammal (e.g, a human).
[0033] The present invention further provides compositions comprising a
therapeutically
active amount of a complex comprising endoxifen and at least one lipid,
wherein said
endoxifen is a free base or is in the form of a salt.
[0034] In some embodiments, the composition comprising endoxifen is formulated
in a
hydroalcoholic gel, a hydroalcoholic solution, a patch, a cream, an emulsion,
a lotion, an
ointment, a powder or an oil.
[0035] In some embodiments, the composition comprising endoxifen is formulated
in a
hydroalcoholic composition containing a penetration enhancer, an aqueous
vehicle, an
alcoholic vehicle and a gelling agent.
[0036] In some embodiments, the hydroalcoholic composition comprises a
neutralizing
agent.
[0037] In some embodibments, the hydroalcoholic composition comprises
endoxifen at
about 0.01 % to 0.20% by weight; isopropyl myristate at about 0.1 % to 2.0%,
preferably
0.5% to 2.0% by weight; alcohol at about 50.0% to 80.0%, preferably about
60.0% to
75.0% by weight; aqueous vehicle at about 20.0% to 60.0%, preferably 25.0% to
50.0% by
weight; and gelling agent at about 1.0% to 10.0%, preferably about 0.5% to
5.0% by
weight. In some embodiments, the wherein the percentage of components is
weight to
weight of the composition.
[0038] The present invention is not limited to the compositions and methods
described
above. It should be understood that variations of the above compositions and
methods
described elsewhere herein or that are understood by a skilled artisan in view
of the present
disclosure are included within the invention.
DESCRIPTION OF THE DRAWINGS
[0039] Figure 1 diagrams compound I.
[0040] Figure 2 diagrams embodiments for synthesis of compounds 3, 5, and I.
[0041] Figure 3 diagrams portions of Tamoxifen metabolic pathways.
DEFINITIONS
[0042] As used herein, the terms "host," "subject" and "patient" refer to any
animal,
including but not limited to, human and non-human animals (e.g., dogs, cats,
cows, horses,
sheep, poultry, fish, crustaceans, etc.) that is studied, analyzed, tested,
diagnosed or treated.
7
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
As used herein, the terms "host," "subject" and "patient" are used
interchangeably, unless
indicated otherwise.
[0043] As used herein, the terms "subject at risk of cancer" refers to a
subject
identified as being at risk for developing cancer, e.g., by prior health
history, genetic data,
etc.
[0044] As used herein, the term "anticancer drug" refers to an agent used to
treat or
prevent cancer. Such agents include, but are not limited to, small molecules,
drugs,
antibodies, pharmaceuticals, and the like.
[0045] As used herein, the term "effective amount" refers to the amount of an
active
composition (e.g., a pharmaceutical compound or composition provided as a
component in
a lipid formulation) sufficient to effect beneficial or desired results. An
effective amount
can be administered in one or more administrations, applications or dosages
and is not
intended to be limited to a particular formulation or administration route.
[0046] As used herein, the terms "active" or "pharmaceutically active" as used
in
reference to an agent, drug, composition, or compound, refers to an agent
that, upon
administration or application, causes a beneficial, desired, or expected
result. The
administration may be in one or more administrations, applications or dosages
and is not
intended to be limited to a particular formulation or administration route.
The term is not
limited to any particular level of activity.
[0047] The terms "agent" and "compound" are used herein interchangeably to
refer
to any atom, molecule, mixture, or more complex composition having an
attributed feature.
For example, an "active agent" or "active compound" refers to any atom,
molecule,
preparation, mixture, etc., that, upon administration or application, causes a
beneficial,
desired, or expected result.
[0048] As used herein, the term "treating" includes administering therapy to
prevent,
cure, or alleviate/prevent the symptoms associated with, a specific disorder,
disease, injury
or condition.
[0049] As used herein, the term "treatment" or grammatical equivalents
encompasses the improvement and/or reversal of the symptoms of disease (e.g.,
cancer), or
reduction of risk of occurrence of disease. A compound which causes an
improvement in
any parameter associated with disease when used in the screening methods of
the instant
invention may thereby be identified as a therapeutic compound. The term
"treatment" refers
to both therapeutic treatment and prophylactic or preventative measures. For
example,
those who may benefit from treatment with compositions and methods of the
present
8
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
invention include those already with a disease and/or disorder (e.g., cancer,
or symptoms or
pathologies consistent with cancer) as well as those in which a disease and/or
disorder is to
be prevented (e.g., using a prophylactic treatment of the present invention).
[0050] As used herein, the term "at risk for disease" refers to a subject
(e.g., a
human) that is predisposed to experiencing a particular disease. This
predisposition may be
genetic (e.g., a particular genetic tendency to experience the disease, such
as heritable
disorders), or due to other factors (e.g., age, weight, environmental
conditions, exposures to
detrimental compounds present in the environment, etc.). Thus, it is not
intended that the
present invention be limited to any particular risk, nor is it intended that
the present
invention be limited to any particular disease.
[0051] As used herein, the term "suffering from disease" refers to a subject
(e.g., a
human) that is experiencing a particular disease. It is not intended that the
present invention
be limited to any particular signs or symptoms, nor disease. Thus, it is
intended that the
present invention encompasses subjects that are experiencing any range of
disease (e.g.,
from sub-clinical manifestation to full-blown disease) wherein the subject
exhibits at least
some of the indicia (e.g., signs and symptoms) associated with the particular
disease.
[0052] As used herein, the terms "disease" and "pathological condition" are
used
interchangeably to describe a state, signs, and/or symptoms that are
associated with any
impairment of the normal state of a living animal or of any of its organs or
tissues that
interrupts or modifies the performance of normal functions, and may be a
response to
environmental factors (such as emotional trauma, physical trauma,
malnutrition, industrial
hazards, or climate), to specific infective agents (such as worms, bacteria,
or viruses), to
inherent defect of the organism (such as various genetic anomalies, or to
combinations of
these and other factors.
[0053] As used herein, the term "administration" refers to the act of giving a
drug,
prodrug, or other active agent, or therapeutic treatment (e.g., compositions
of the present
invention) to a physiological system (e.g., a subject or in vivo, in vitro, or
ex vivo cells,
tissues, and organs). Exemplary routes of administration to the human body can
be through
the eyes (ophthalmic), mouth (oral), skin (transdermal), nose (nasal), lungs
(inhalant),
rectal, vaginal, oral mucosa (buccal), ear, by injection (e.g., intravenously,
subcutaneously,
intratumorally, intraperitoneally, etc.) and the like. Administration may be
in one or more
administrations, applications or dosages, and is not intended to be limited to
a particular
administration route.
9
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
[0054] As used herein, the term "co-administration" refers to the
administration of at
least two agent(s) (e.g., two separate lipid compositions, containing
different active
compounds) or therapies to a subject. In some embodiments, the co-
administration of two
or more agents or therapies is concurrent. In other embodiments, a first
agent/therapy is
administered prior to a second agent/therapy. Those of skill in the art
understand that the
formulations and/or routes of administration of the various agents or
therapies used may
vary. The appropriate dosage for co-administration can be readily determined
by one
skilled in the art. In some embodiments, when agents or therapies are co-
administered, the
respective agents or therapies are administered at lower dosages than
appropriate for their
administration alone. Thus, co-administration is especially desirable in
embodiments where
the co-administration of the agents or therapies lowers the requisite dosage
of a potentially
harmful (e.g., toxic) agent(s).
[0055] As used herein, the term "toxic" refers to any detrimental or harmful
effects
on a subject, a cell, or a tissue as compared to the same cell or tissue prior
to the
administration of the toxicant.
[0056] As used herein, the term "pharmaceutically purified" refers to a
composition
of sufficient purity or quality of preparation for pharmaceutical use.
[0057] As used herein, the term "purified" refers to a treatment of a starting
composition to remove at least one other component (e.g., another component
from a
starting composition (e.g., plant or animal tissue, an environmental sample
etc.), a
contaminant, a synthesis precursor, or a byproduct, etc.), such that the ratio
of the purified
component to the removed component is greater than in the starting
composition.
[0058] As used herein, the term "pharmaceutical composition" refers to the
combination of an active agent (e.g., an active pharmaceutical compound) with
a carrier,
inert or active (e.g., a phospholipid), making the composition especially
suitable for
diagnostic or therapeutic use in vitro, in vivo or ex vivo.
[0059] The terms "pharmaceutically acceptable" or "pharmacologically
acceptable,"
as used herein, refer to compositions that do not substantially produce
adverse reactions,
e.g., toxic, allergic, or immunological reactions, when administered to a
subject.
[0060] As used herein, the term "topically" refers to application of the
compositions
of the present invention to the surface of the skin and mucosal cells and
tissues (e.g.,
alveolar, buccal, lingual, masticatory, or nasal mucosa, and other tissues and
cells which
line hollow organs or body cavities).
CA 02669913 2011-09-15
[0061] As used herein, the term "pharmaceutically acceptable carrier" refers
to any
of the standard pharmaceutical carriers including, but not limited to,
phosphate buffered
saline solution, water, emulsions (e.g., such as an oil/water or water/oil
emulsions), and
various types of wetting agents, any and all solvents, dispersion media,
coatings, sodium
lauryl sulfate, isotonic and absorption delaying agents, disintigrants (e.g.,
potato starch or
sodium starch glycolate), and the like.. The compositions also can include
stabilizers and
preservatives. For examples of carriers, stabilizers, and adjuvants. (See
e.g., Martin,
Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa.
(1975) ).
Moreover, in certain embodiments, the compositions of
the present invention may be formulated for horticultural or agricultural use.
Such
formulations include dips, sprays, seed dressings, stem injections, sprays,
and mists.
[0062] As used herein, the term "pharmaceutically acceptable salt" refers to
any salt
(e.g., obtained by reaction with an acid or a base) of a compound of the
present invention
that is physiologically tolerated in the target subject (e.g., a mammalian
subject, and/or in
vivo or ex vivo, cells, tissues, or organs). "Salts" of the compounds of the
present invention
may be derived from inorganic or organic acids and bases. Examples of acids
include, but
are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric,
fumaric, maleic,
phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic,
tartaric, acetic, citric,
methanesulfonic, ethanesulfonic, formic, benzoic, malonic, sulfonic,
naphthalene-2-
sulfonic, benzenesulfonic acid, and the like. Other acids, such as oxalic,
while not in
themselves pharmaceutically acceptable, may be employed in the preparation of
salts useful
as intermediates in obtaining the compounds of the invention and their
pharmaceutically
acceptable acid addition salts.
[0063] Examples of bases include, but are not limited to, alkali metal (e.g.,
sodium)
hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and
compounds
of formula NW4+, wherein W is C1_4 alkyl, and the like.
[0064] Examples of salts include, but are not limited to: acetate, adipate,
alginate,
aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate,
camphorate,
camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate,
fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate,
hexanoate, chloride,
bromide, iodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,
2-
naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate,
phenylpropionate,
picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate,
undecanoate, and the
like. Other examples of salts include anions of the compounds of the present
invention
11
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
compounded with a suitable cation such as Na-'-, NH4, and NW4+ (wherein W is a
Ci_4 alkyl
group), and the like. For therapeutic use, salts of the compounds of the
present invention
are contemplated as being pharmaceutically acceptable. However, salts of acids
and bases
that are non-pharmaceutically acceptable may also find use, for example, in
the preparation
or purification of a pharmaceutically acceptable compound.
[0065] For therapeutic use, salts of the compounds of the present invention
are
contemplated as being pharmaceutically acceptable. However, salts of acids and
bases that
are non-pharmaceutically acceptable may also find use, for example, in the
preparation or
purification of a pharmaceutically acceptable compound.
[0066] As used herein, the term "hydroalcoholic" as used in reference to a
substance
or composition indicates that said substance or composition comprises both
water and
alcohol.
[0067] As used herein, the term "gelling agent" refers to a composition that,
when
dissolved, suspended or dispersed in a fluid (e.g., an aqueous fluid such as
water or a buffer
solution), forms a gelatinous semi-solid (e.g., a lubricant gel). Examples of
gelling agents
include but are not limited to hydroxyethyl cellulose, hydroxymethyl
cellulose,
hydroxypropyl guar, methyl cellulose, ethyl cellulose, hydroxypropyl
cellulose, sodium
carboxymethyl cellulose, carbomer, alginate, gelatin, and poloxamer.
[0068] As used herein, the term "dried" as used in reference to a composition
refers
to removing the solvent component or components to levels that no longer
support chemical
reactions. The term is also used in reference to a composition that has been
dried (e.g., a
dried preparation or dried composition). Those of skill in the art will
appreciate that a
composition may be "dried" while still having residual solvent or moisture
content after,
e.g., lyophilization, or that a dried composition may, after the end of a
drying process,
absorb moisture hygroscopically, e.g., from the atmosphere. The term "dried"
encompasses
a composition with increased moisture content due to hygroscopic absorption.
[0069] As used herein, the term "protective agent" refers to a composition or
compound that protects the activity or integrity of an active agent (e.g., an
anticancer drug)
when the active agent is exposed to certain conditions (e.g., drying,
freezing). In some
embodiments, a protective agent protects an active agent during a freezing
process (i.e., it is
a "cryoprotectant"). Examples of protective agents include but are not limited
to non-fat
milk solids, trehalose, glycerol, betaine, sucrose, glucose, lactose, dextran,
polyethylene
glycol, sorbitol, mannitol, poly vinyl propylene, potassium glutamate,
monosodium
glutamate, Tween 20 detergent, Tween 80 detergent, and an amino acid
hydrochloride.
12
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
[0070] As used herein, the term "excipient" refers to an inactive ingredient
(i.e.,
not pharmaceutically active) added to a preparation of an active ingredient.
The gelling and
protective agents described herein may be referred to generally as
"excipients."
[0071] As used herein, the term "kit" refers to any delivery system for
delivering
materials. In the context of kinase activity or inhibition assays, such
delivery systems
include systems that allow for the storage, transport, or delivery of reaction
reagents and/or
supporting materials (e.g., buffers, written instructions for performing the
assay etc.) from
one location to another. For example, kits include one or more enclosures
(e.g., boxes)
containing the relevant reaction reagents and/or supporting materials. As used
herein, the
term "fragmented kit" refers to delivery systems comprising two or more
separate
containers that each contains a subportion of the total kit components. The
containers may
be delivered to the intended recipient together or separately. For example, a
first container
may contain an agent for use in an assay, while a second container contains
standards for
comparison to test compounds. The term "fragmented kit" is intended to
encompass kits
containing Analyte Specific Reagents (ASR's) regulated under section 520(e) of
the Federal
Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any
delivery system
comprising two or more separate containers that each contains a subportion of
the total kit
components are included in the term "fragmented kit." In contrast, a "combined
kit" refers
to a delivery system containing all of the components of a reaction assay in a
single
container (e.g., in a single box housing each of the desired components). The
term "kit"
includes both fragmented and combined kits.
DETAILED DESCRIPTION OF THE INVENTION
[0072] The present invention provides compositions and methods for delivering
endoxifen of Formula I, e.g., to a mammalian host. In some embodiments of the
present
invention endoxifen is an E-isomer, while in other embodiments, it is a Z-
isomer, while it is
still in other embodiments, it is a mixture of E- and Z-isomers
,C H3
O __/-N'H
I~ ~I
H3CH2C
OH
1
13
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
[0073] An example of the present invention includes endoxifen, analogues of
endoxifen,
and derivatives of endoxifen, including but not limited to endoxifen,
Tamoxifen, and 4-
hydroxytamoxifen. The present invention also find use with other
antineoplastic agents
such as paclitaxel, docetaxel, melphalan, chlormethine, extramustinephosphate,
uramustine,
ifosfamide, mannomustine, trifosfamide, streptozotocin, mitobronitol,
mitoxantrone,
methotrexate, fluorouracil, cytarabine, tegafur, idoxide, taxol, paclitaxel,
daunomycin,
daunorubicin, bleomycin, amphotericin, carboplatin, cisplatin, BCNU,
vincristine,
camptothecin, SN-38, doxorubicin, and etopside. Also included are steroidal
and non-
steroidal inhibitors used in cancer treatment, such as bicautamide,
exemestane, formestane,
letrozole, anastrazole and their analogues.
[0074] Endoxifen of Formula I can be prepared by any desired method for use in
the
treatments of the present invention but, in some embodiments, the present
invention
provides particular methods for the preparation of endoxifen. One preferred
method of the
present invention is set forth in Figure 2. In this method, 4-bromophenol 1 is
reacted with
3,4-dihydropyran 2 in the present of acid (e.g., sulfuric acid and the like),
to give compound
3. Compound 3 is then reacted with magnesium turning in a suitable anhydrous
solvent
(e.g., tetrahydrofuran and the like). This is followed by reaction with 1-[4-
(2-
chloroethoxy)phenyl]-2-phenyl-l-butanone 4 to provide compound 5 which, on
dehydration/deprotection in presence of acid in a suitable solvent (e.g.,
methanol and the
like), produces compound 6. Reaction of yielded compound 6 with methylamine in
a
suitable solvent (e.g., isopropanol and the like) provides endoxifen I.
[0075] In some embodiments of the present invention, a mixture of E- and Z-
isomers of
endoxifen can be separated to provide the purified preparations of E- and Z-
isomer of
endoxifen. The separation of E- and Z- isomers of endoxifen in the present
invention can be
done, e.g., by crystallization, or purification by liquid column
chromatography (LC), or high
pressure liquid column chromatography (HPLC).
[0076] Suitable solvents that can be employed in present invention for the
separation of
E- and Z-isomers of endoxifen include but are not limited to hexanes,
heptanes, and the like,
benzene; toluene; ethyl acetate; acetonitrile; chlorinating solvents such as
methylene
chloride, chloroform, 1,2-dichloromethane, and the like, ketones, (e.g.,
acetone, 2-
butanone, and the like), ethers such as diethyl ether, diisopropyl ether,
methyl butyl ether,
and tetrahydrofuran, alcohols such as methanol, ethyl alcohol, and isopropyl
alcohol, and
the like, and water. A solvent for crystallization can be used as a single
solvent, or as
14
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
mixture of solvents such as hexane-ethyl acetate, chloroform-acetone,
chloroform-
methanol, dichloromethane-methanol, and the like. When a mixture of two
solvents is used
in the present invention, examples of ratios of one solvent to another are
e.g., in a range
such as 9:1 to 1:9, (e.g., 8:2, 7:3; 6:4; 5:5; 4:6; 3:7; 2:8; 1:9, and the
like.) However,
mixtures for use in the present invention are not limited to these ratios, or
to mixtures
comprising only two solvents.
[0077] Solvents that find use in the preparation of endoxifen according to the
present
invention include but are not limited to tetrahydrofuran, dichloromethane,
chloroform, 1,2-
dichloroethane, acetonitrile, N,N'-dimethylformamide, dimethylsulfoxide,
toluene, pyridine,
methanol, ethanol, isopropanol, acetone, 2-butanone, hexane, heptane, pentane,
ethyl
acetate, and the like.
[0078] Acids that find use in the preparation of endoxifen according to the
present
invention include, but are not limited to, sulfuric acid, hydrochloric acid,
acetic acid,
trifluroacetic acid, phosphoric acid, p-toluenesulfonic acid, methanesulfonic
acid, nitric
acid, and the like.
[0079] Intermediates and final products of the present invention can be
purified by
column chromatography using a single or a mixture of common organic solvents
such as
hexane pentane, heptane, ethyl acetate, methylene chloride, chloroform,
methanol, acetone,
and the like.
[0080] As noted above, intermediates and final product of the present
invention, may, in
some embodiments, be purified by crystallization. Solvents that find use in
the
crystallization of intermediates and products include but are not limited to
hydrocarbons
such as pentanes, hexanes, heptanes, and the like, benzene; toluene; ethyl
acetate;
acetonitrile; chlorinating solvents such as methylene chloride, chloroform,
1,2-
dichloromethane, and the like; ketones, for example, acetone, 2-butanone, and
the like;
ethers such as diethyl ether, diisopropyl ether, methyl butyl ether,
tetrahydrofuran; alcohols
such as methanol, ethyl alcohol, isopropyl alcohol, and the like. A solvent
for
crystallization can be used as a single solvent or mixture of solvents.
Exemplary mixtures
include, e.g., hexane-ethyl acetate, chloroform-acetone, chloroform-methanol,
dichloromethane-methanol, and the like. When a mixture of two solvents is used
in the
present invention, examples of ratios of one solvent to another are e.g., in a
range such as
9:1 to 1:9, (e.g., 8:2, 7:3; 6:4; 5:5; 4:6; 3:7; 2:8; 1:9, and the like.)
However, mixtures for
use in the present invention are not limited to these ratios, or to mixtures
comprising only
two solvents.
CA 02669913 2011-09-15
[0081] One object of the present invention is to provide E-endoxifen or Z-
endoxifen with
at least 80% purity, such as at least 90% pure or at least 95% pure or at
least 98% pure or at
least 99% pure or at least 100% pure.
[0082] Another object of the present invention is to provide solubilized
endoxifen in,
e.g., aqueous acid. Suitable acids for solubilizing endoxifen include but are
not limited to
formic acid, acetic acid, propionic acid, butyric acid, trifuloroacetic acid,
lactic acid, tartaric
acid, oxalic acid, malonic acid, succinic acid, and the like. The pH of the
acidic solution
comprising endoxifen can be adjusted with suitable base or buffers. Examples
of base and
buffers include but are not limited to sodium hydroxide, sodium acetate,
sodium lactate,
sodium succinate, sodium monophosphate, sodium diphosphate, sodium
triphosphate,
sodium oxalate, sodium tartarate, ammonium hydroxide, ammonium acetate, and
the like.
In some embodiments, a co-solvent can also be used to solubilize endoxifen.
Examples of
co-solvent include but are not limited to ethanol, isopropanol, detergents
such as Tween 20TM
and Polysorbate, and the like
[0083] In certain preferred embodiments, the pH of a composition containing
endoxifen
according to the present invention are between about 4.0 and about 8.0, and
preferably
between about 5.0 and about 8.0, and most preferably between about 5.5 and
about7.5.
[0084] In some embodiments, the present invention relates to compositions and
methods
for delivery of endoxifen or endoxifen-lipid complexes to a mammalian host.
Any suitable
amount of endoxifen can be used in complex formation. Suitable amounts of
endoxifen are
those amounts that can be stably incorporated into the complexes of the
present invention.
[0085] In some embodiments, the inventive composition comprises a lipid
complex with
endoxifen in which the complex desirably contains lipid or a mixture of
lipids. Complexes
can be in the form, e.g., of micelles, vesicles or emulsions without exclusion
of other forms.
The micelles of the present invention can be in the form of monomeric,
dimeric, polymeric
or mixed micelles. The complexes including micelles and emulsions are
predominately in
the size range of 50nm-20 micron, preferably in size range of 50nm-5 micron.
In the
complexes, the active agent can be bound to the lipid by covalent,
hydrophobic,
electrostatic, hydrogen, or other bonds, and is considered bound even where
the drug is
simply entrapped within the interior of lipid structures.
[0086] Endoxifen-lipid complexes may contain e.g., cholesterols or cholesterol
derivatives or a mixture of cholesterol and cholesterol derivatives.
Cholesterol derivatives
that find use in the present invention include cholesteryl hemisuccinate,
cholesteryl
succinate, cholesteryl oleate, cholesteryl linoleate, cholesteryl
eicosapentenoate, cholesteryl
16
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
linolenate, cholesteryl arachidonate, cholesteryl palmitate, cholesteryl
stearate, cholesteryl
myristate, polyethylene glycol derivatives of cholesterol (cholesterol-PEG),
water soluble
cholesterol (for example, cholesterol methyl-(3-cyclodextrin), coprostanol,
cholestanol, or
cholestane, cholic acid, cortisol, corticosterone or hydrocortisone and 7-
dehydrocholesterol.
[0087] In some preferred embodiments, the compositions also include a-, (3-, y-
tocopherols, vitamin E, calciferol, organic acid derivatives of a-, (3-, y-
tocopherols, such as
a-tocopherol hemisuccinate (THS), a-tocopherol succinate and/or mixtures
thereof.
[0088] In other some preferred embodiments, endoxifen-lipid complexes of the
present
invention contain sterols. Sterols that find use in the present invention
include 0-sitosterol,
stigmasterol, stigmastanol, lanosterol, a-spinasterol, lathosterol,
campesterol and/or
mixtures thereof.
[0089] Compositions of the present invention also include endoxifen complexes
with
free and/or salts or esters of fatty acid. Preferred fatty acids range from
those with carbon
chain lengths of about C2 to C34, preferably between about C4 and about C24,
and include
tetranoic acid (C4:0), pentanoic acid (C5:0), hexanoic acid (C6:0), heptanoic
acid (C7:0),
octanoic acid (C8:0), nonanoic acid (C9:0), decanoic acid (C10:0), undecanoic
acid (C11:0),
dodecanoic acid (C12:0), tridecanoic acid (C13:0), tetradecanoic (myristic)
acid (C14:0),
pentadecanoic acid (C15:0), hexadecanoic (palmatic) acid (C16:0),
heptadecanoic acid (C17:0),
octadecanoic (stearic) acid (C18:0), nonadecanoic acid (C19:0), eicosanoic
(arachidic) acid
(C20:0), heneicosanoic acid (C21:0), docosanoic (behenic) acid (C22:0),
tricosanoic acid (C23:0),
tetracosanoic acid (C24:0), l0-undecenoic acid (C11:1), 11-dodecenoic acid
(C12:1), 12-
tridecenoic acid (C13:1), myristoleic acid (C14:1), l0-pentadecenoic acid
(C15:1), palmitoleic
acid (C16:i), oleic acid (C1g:1), linoleic acid (C18:2), linolenic acid
(C18:3), eicosenoic acid
(C20:i), eicosdienoic acid (C20:2), eicosatrienoic acid (C20:3), arachidonic
acid (cis-5,8,11,14-
eicosatetraenoic acid), and cis-5,8,11,14,17-eicosapentaenoic acid, among
others. Other
fatty acids also can be employed in the compositions. Examples of such include
saturated
fatty acids such as ethanoic (or acetic) acid, propanoic (or propionic) acid,
butanoic (or
butyric) acid, hexacosanoic (or cerotic) acid, octacosanoic (or montanic)
acid, triacontanoic
(or melissic) acid, dotriacontanoic (or lacceroic) acid, tetratriacontanoic
(or gheddic) acid,
pentatriacontanoic (or ceroplastic) acid, and the like; monoethenoic
unsaturated fatty acids
such as trans-2-butenoic (or crotonic) acid, cis-2-butenoic (or isocrotonoic)
acid, 2-
hexenoic (or isohydrosorbic) acid, 4-decanoic (or obtusilic) acid, 9-decanoic
(or caproleic)
acid, 4-dodecenoic (or linderic) acid, 5-dodecenoic (or denticetic) acid, 9-
dodecenoic (or
lauroleic) acid, 4-tetradecenoic (or tsuzuic) acid, 5-tetradecenoic (or
physeteric) acid, 6-
17
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
octadecenoic (or petroselenic) acid, trans-9-octadecenoic (or elaidic) acid,
trans- ll-
octadecenoic (or vaccinic) acid, 9-eicosenoic (or gadoleic) acid, 11-
eicosenoic (or
gondoic) acid, 11-docosenoic (or cetoleic) acid, 13-decosenoic (or erucic)
acid, 15-
tetracosenoic (or nervonic) acid, 17-hexacosenoic (or ximenic) acid, 2 1 -
triacontenoic ( or
lumequeic) acid, and the like; dienoic unsaturated fatty acids such as 2,4-
pentadienoic (or
(3-vinylacrylic) acid, 2,4-hexadienoic (or sorbic) acid, 2,4-decadienoic (or
stillingic) acid,
2,4-dodecadienoic acid, 9,12-hexadecadienoic acid, cis-9, cis-12-
octadecadienoic (or a-
linoleic) acid, trans-9, trans- l2-octadecadienoic (or linlolelaidic) acid,
trans- I O,trans- 12-
octadecadienoic acid, 11,14-eicosadienoic acid, 13,16-docosadienoic acid,
17,20-
hexacosadienoic acid and the like; trienoic unsaturated fatty acids such as
6,10,14-
hexadecatrienoic (or hiragonic) acid, 7,10,13-hexadecatrienoic acid, cis-6,
cis-9- cis-12-
octadecatrienoic (or y-linoleic) acid, trans-8, trans-10- trans-l2-
octadecatrienoic (or J3-
calendic) acid, cis-8, trans-10- cis- l2-octadecatrienoic acid, cis-9, cis-12-
cis-15-
octadecatrienoic (or a-linolenic) acid, trans-9, trans-12- trans- l5-
octadecatrienoic (or a-
linolenelaidic) acid, cis-9, trans- ll- trans- l3-octadecatrienoic (or a-
eleostearic) acid, trans-
9, trans-11- trans- l3-octadecatrienoic (or (3-eleostearic) acid, cis-9, trans-
11- cis- 13-
octadecatrienoic (or punicic) acid, 5,8, 11 -eicosatrienoic acid, 8,11,14-
eicosatrienoic acid
and the like; tetraenoic unsaturated fatty acids such as 4,8,11,14-
hexadecatetraenoic acid,
6,9,12,15- hexadecatetraenoic acid, 4,8,12,15-octadecatetraenoic (or moroctic)
acid,
6,9,12,15- octadecatetraenoic acid, 9,11,13,15- octadecatetraenoic (or a -or
(3-parinaric)
acid, 9,12,15,18-octadecatetraenoic acid, 4, 8,12,16-eicosatetraenoic acid,
6,10,14,18-
eicosatetraenoic acid, 4,7,10,13-docasatetraenoic acid, 7,10,13,16-
docosatetraenoic acid,
8,12,16,19-docosatetraenoic acid and the like; penta- and hexa-enoic
unsaturated fatty acids
such as 4,8,12,15,18-eicosapentaenoic (or timnodonic) acid, 4,7,10,13,16-
docosapentaenoic
acid, 4,8,12,15,19-docosapentaenoic (or clupanodonic) acid, 7,10,13,16,19-
docosapentaenoic, 4,7,10, 13,16,19-docosahexaenoic acid, 4,8,12,15,18,21-
tetracosahexaenoic (or nisinic) acid and the like; branched-chain fatty acids
such as 3-
methylbutanoic (or isovaleric) acid, 8-methyldodecanoic acid, 10-
methylundecanoic (or
isolauric) acid, 11-methyldodecanoic (or isoundecylic) acid, 12-
methyltridecanoic (or
isomyristic) acid, 13-methyltetradecanoic (or isopentadecylic) acid, 14-
methylpentadecanoic (or isopalmitic) acid, 15-methylhexadecanoic, 10-
methylheptadecanoic acid, 16-methylheptadecanoic (or isostearic) acid, 18-
methylnonadecanoic (or isoarachidic) acid, 20-methylheneicosanoic (or
isobehenic) acid,
22-methyltricosanoic (or isolignoceric) acid, 24-methylpentacosanoic (or
isocerotic) acid,
18
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
26-methylheptacosanoic (or isomonatonic) acid, 2,4,6-trimethyloctacosanoic (or
mycoceranic or mycoserosic) acid, 2-methyl-cis-2-butenoic(angelic)acid, 2-
methyl-trans-2-
butenoic (or tiglic) acid, 4-methyl-3-pentenoic (or pyroterebic) acid and the
like.
[0090] In some preferred embodiments, endoxifen-lipid complexes contain
phospholipids. Any suitable phospholipids or mixture of phospholipids can be
used. For
example, phospholipids can be obtained from natural sources or chemically
synthesized.
Suitable phospholipids include but are not limited to phosphatidylethanolamine
(PE),
phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylcholine (PC),
phosphatidylinositol (PI), phosphatidic acid (PA), sphingomyelin and the like,
either used
separately or in combination. Phosphatidylglycerols may be having short chain
or long
chain, saturated or unsaturated such as dimyristoylphosphatidylglycerol,
dioleoylphosphatidylglycerol, distearoylphosphatidylglycerol,
dipalmitoylphosphatidylglycerol, diarachidonoylphosphatidylglycerol, short
chain
phosphatidylglycerol (C6-Cg), and mixtures thereof. Examples of
phosphatidylcholines
includes dimyristoylphophatidylcholine, distearoylphosphatidylcholine,
dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine,
diarachidonoylphosphatidylcholine, egg phosphatidylcholine, soy
phosphatidylcholine or
hydrogenated soy phosphatidylcholine can be used, as can mixtures thereof.
[0091] According to one aspect, the present invention provides compositions
comprising
endoxifen and derivatives of mono-, di- and tri-glycerides. Examples of the
glycerides
include 1-oleoyl-glycerol (monoolein) and 1, 2-dioctanoyl-sn-glycerol.
[0092] Another aspect of the invention provides forming complexes of endoxifen
with
functionalized phospholipids including but not limited to
phosphatidylethanolamine,
preferably dioleoylphosphatidylethanolamine, phosphatidylthioethanol, N-
biotinylphosphatidylethanolamine and phosphatidylethylene glycol.
[0093] Another aspect of the invention provides forming complexes of endoxifen
with
carbohydrate-based lipids. Examples of carbohydrate-based lipids include but
are not
limited to galactolipids, mannolipids, galactolecithin and the like.
[0094] In other preferred embodiment, endoxifen-lipid complexes comprise
sterols.
Sterols finding use in the present invention include but are not limited to 0-
sitosterol,
stigmasterol, stigmastanol, lanosterol, a-spinasterol, lathosterol,
campesterol and/or
mixtures thereof.
19
CA 02669913 2011-09-15
[0095] Another aspect of the invention provides forming complexes of endoxifen
with
guggulipid and any suitable phospholipids. Guggulipid, or guggul, is a natural
substance
derived from the mukul myrrh tree. The mukul myrrh gives off a sticky resin,
which is
processed to obtain guggulipid. This extract has been used for thousands of
years in
Aryuvedic medicine to treat arthritis and obesity. The guggulipid is a source
of sterol
compounds such as Z- and E-guggulsterones, generally present in an amount of
at least
2.5% (10). Z and E-Guggulsterones can be synthesized chemically and thus can
be used in
drug formulations where the need is to have pure forms of these sterones. See,
e.g.,
International Publication No. WO/2008/058156, filed November 6, 2007.
[0096] Yet another aspect of the invention provides forming complexes of
endoxifen
with derivatives of phospholipids such as pegylated phospholipids. Examples of
pegylated
lipids finding use in the present invention include but are not limited to the
polyethylene
glycol (Pegylated, PEG) derivatives of distearoylphosphatidylglycerol,
dimyristoylphosphatidylglycerol, dioleoylphosphatidylglycerol and the like.
[0097] In other aspects, the present invention provides compositions
comprising
endoxifen and polyethyleneglycol (PEG) and one or more lipids.
[0098] According to yet other aspects, the present invention provides
compositions
comprising endoxifen complexes with one or more lipids. Examples include but
are not
limited to compositions comprising endoxifen, cholesterol or cholesterol
derivatives and
one or more phospholipids. Other examples of compositions include endoxifen,13-
sitosterol, and one or more phospholipids. In some preferred embodiments,
compositions of
the present invention comprise endoxifen, and hydrogenated soy
phosphatidylcholine or soy
phosphatidylcholine.
[0099] The term "Polyethylene glycol (PEG)" includes polymers of lower
alkylene
oxide, in particular ethylene oxide (polyethylene glycols) having an
esterifiable hydroxyl.
group at least at one end of the polymer molecule, as well as derivatives of
such polymers
having esterifiable carboxy groups. Polyethylene glycols of an average
molecular weight
ranging from 200-20,000 are preferred; those having an average molecular
weight ranging
from 500-2000 are particularly preferred.
[00100] Another aspect of the invention provides forming complexes of
endoxifen
with carbohydrate-based lipids. Examples of carbohydrate based lipids include
but are not
limited to galactolipids, mannolipids, galactolecithin and the like.
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
[00101] In some embodiments of compositions of the invention, a complex is
formed
comprising endoxifen and preferably endoxifen in water at a concentration of
about 0.5
mg/mL to about 25 mg/mL, such as between 1 mg/mL and about 20 mg/mL or between
1
mg/mL and 10 mg/mL, more preferably between 1 mg/mL and 5 mg/mL.
[00102] In some embodiments, compositions of the present invention contain
about
2.5% to about 90% of total lipid, preferably about 2.5 to about 50% weight of
total lipid or
more, preferably about 10% to about 50% weight of total lipid.
[00103] In certain embodiments, compositions of the present invention
preferably
contain endoxifen, and lipid(s) in mole ratio between 1:1 to 1:100 such as in
between 1:1
and 1:20 molar ratio or in between 1:1 and 1:30 molar ratio or in between 1:1
and 1:40
molar ratio or in between 1:1 and 1:50 molar ratio, in between 1:1 and 1:60
molar ratio, in
between 1:1 and 1:70 molar ratios, and in between 1:1 and 1:80 molar ratios,
and 1:90
molar ratios.
[00104] Ratios recited herein, e.g., mole ratios of components in a
composition, are
provided by way of example and do not limit the invention to the precise
incremental ratios
recited, e.g., to whole number ratios of the components in the composition.
For example, a
range of ratios of about 1:10 to 1:90 encompasses not only 1:11, 1:25, 1:89,
etc., but
includes, without limitation, any ratio at or between about 1:10 to 1:90
(e.g., 1:53.637).
[00105] In certain embodiments, compositions of the present invention
preferably
contain endoxifen and hydrogenated soy phosphatidylcholine, or soy
phosphatidylcholine,
and cholesterol or cholesterol derivative. Such composition includes endoxifen
and
cholesterol or cholesterol derivative preferably in from about 1:1-1:5 mole
ratio, and more
preferably at about 1:1 mole ratio to about 1:2 mole ratio.
[00106] Yet another aspect of the invention is to form complexes of endoxifen
with
derivatives of phospholipids, such as pegylated phospholipids. Examples
include but are
not limited to the polyethylene glycol (PEG) derivatives of
distearoylphosphatidylglycerol,
dimyristoylphosphatidylglycerol, dioleoylphosphatidylglycerol and the like.
[00107] In some preferred embodiments, the mole ratio of endoxifen and
hydrogenated soy phosphatidylcholine or soy phosphatidylcholine, in a
composition
containing endoxifen and hydrogenated soy phosphatidylcholine or
phosphatidylcholine is
between about 1:10 and 1:90, e.g., between about 1:10 and 1:80 orl:10 and 1:70
or 1:10
and 1:60 or 1:10 and 1:50 or 1:10 and 1:40 and 1:10 and 1:30. Particularly
preferred
embodiments, the mole ratio of endoxifen and hydrogenated soy
phosphatidylcholine or soy
phosphatidylcholine is between 1:10 and 1:60.
21
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
[00108] In some embodiments, compositions of the present invention preferably
contain endoxifen and total lipids having weight to weight ratio between 1:1
to 1:100 ratio
such as between 1:1 and 1:20 ratio or between 1:1 and 1:30 ratio or between
1:1 and 1:40
ratio or between 1:1 and 1:50 ratio, or between 1:1 and 1:60 ratio, or between
1:1 and 1:70
ratio, and between 1:1 and 1:80 ratio, or in between 1:1 and 1:90 ratio.
[00109] In some embodiments, the method of the present invention comprises
solubilizing or suspending endoxifen and lipid(s) together in an aqueous
solution, e.g.,
water. Endoxifen-lipid complex solution can be filtered through suitable
filters to control
the size distribution of the complexes.
[00110] In some embodiments, the method may comprise mixing lipid(s) together
in
water and then adding endoxifen. Endoxifen-lipid complex solution can be
filtered through
suitable filters to control the size distribution of the complexes.
[00111] In some embodiments, the method also comprises mixing endoxifen and
lipid(s) in an organic solvent(s), such as chloroform or ethanol or any other
pharmaceutically acceptable solvents, and evaporating the solvent(s) to form a
lipid phase
or lipid film. The lipid phase is then hydrated with water or an aqueous
solution. Examples
of aqueous solutions include but are not limited to 0.9% sodium chloride,
solutions
containing sugars such as dextrose, sucrose, and the like. The hydrated
solution can be
filtered through suitable filters to control the size distribution of the
complexes.
[00112] In some embodiments, the method comprises mixing lipid(s) in an
organic
solvent(s) and evaporating the solvent(s) to form a lipid phase or lipid film.
The lipid phase
is then hydrated with aqueous solution containing endoxifen. The aqueous
solution in
addition to endoxifen may further contain sodium chloride or sugars such as
dextrose,
sucrose and the like. The hydrated solution can be filtered through suitable
filters to control
the size distribution of the complexes.
[00113] In other embodiments, the method of the present invention comprises
mixing
endoxifen, one or more lipids in any suitable order and in any suitable
solvents such that the
resulting composition of the present invention contains endoxifen, and one or
more lipids.
[00114] In some embodiments, the method of preparation of the present
invention
comprises heating the composition comprising endoxifen, and the lipid(s) at
temperatures
ranging from 30-100 C preferably between 30 - 80 C and more preferably
between 30-
60 C.
22
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
[00115] In some embodiments, the pH of the composition of invention ranges
from
about 3 to about 11, while a pH between3.5 to about 8 is preferred and pH of
between 4.0 to
pH 7.5 are particularly preferred. Aqueous solutions having of a particular pH
can be
prepared from water having comprising appropriate buffers. Preferred buffers
include but
are not limited to mixtures of monobasic sodium phosphate and dibasic sodium
phosphate,
tribasic sodium phosphate, disodium succinate. Other buffers that find use
with the present
invention include sodium carbonate, sodium bicarbonate, sodium hydroxide,
ammonium
acetate, sodium citrate, tris (hydroxy-methyl) aminoethane, sodium benzoate,
and the like.
[00116] The mole ratio of endoxifen and hydrogenated soy phosphatidylcholine
or
soy phosphatidylcholine in the composition containing endoxifen and
hydrogenated soy
phosphatidylcholine or soy phosphatidylcholine is in between 1:10 and 1:90
such as in
between 1:10 and 1:80 or 1:10 and 1:80 or 1:10 and 1:60 or 1:10 and 1:50 or
1:10 and 1:40
and 1:10 and 1:30. In preferred embodiments, the mole ratio of endoxifen and
hydrogenated soy phosphatidylcholine or soy phosphatidylcholine is in between
1:5 and
1:60.
[00117] As noted above, compositions can be filtered to obtain a desired size
range of
complexes particle sizes from the filtrate. Filters that find use in the
present invention
include those that can be used to obtain the desired size range of the
complexes from the
filtrate. For example, the complexes can be formed and thereafter filtered
through a 5
micron filter to obtain complexes, each particle having a diameter of about 5
micron or less.
Alternatively, 1 m, 500 nm, 200 nm, 100 nm or other filters can be used to
obtain
complexes having diameters of about 1 m, 500 nm, 200 nm, 100 nm or any
suitable size
range, respectively.
[00118] When desired, the endoxifen-lipid complex can be dried, e.g., by
evaporation
or lyophilization. In certain embodiments of the invention, the endoxifen-
lipid complex can
be lyophilized with one or more cryoprotectants such as sugars. In preferred
embodiments,
sugars include but are not limited to trehalose, maltose, lactose, sucrose,
glucose, and
dextran. In particularly preferred embodiments, trehalose and/or sucrose are
used.
Lyophilization is accomplished under vacuum and can take place either with or
without
prior freezing of the endoxifen lipid preparation. When desired, the complexes
can be
resuspended in any desirable solvent including water, saline, dextrose and
buffer.
[00119] Pharmaceutical preparations that find use with the compositions of the
present invention include but are not limited to tablets, capsules, pills,
dragees,
suppositories, solutions, suspensions, emulsions, ointments, and gels. For the
oral mode of
23
CA 02669913 2011-09-15
administration, preferred forms of endoxifen or endoxifen lipid complex
include tablets,
capsules, lozenges, powders, syrups, aqueous solutions, suspensions and the
like. For
topical application and suppositories, preferred forms of endoxifen or
endoxifen-lipid
complex comprise gels, oils, and emulsions, such as are formed by the addition
of suitable
water-soluble or water-insoluble excipients, for example polyethylene glycols,
certain fats,
and esters, compounds having a higher content of polyunsaturated fatty acids
and
derivatives thereof. Derivatives include mono-, di-, and triglycerides and
their aliphatic
esters (for example, fish oils, vegetable oils etc.) or mixtures of these
substances. Suitable
excipients are those in which the drug complexes are sufficiently stable to
allow for
therapeutic use.
[00120] When desired, composition containing endoxifen or endoxifen-lipid
complex can be encapsulated in enteric-coated capsules to protect it from
acids in the
stomach. The term "enteric" refers to the small intestine, and enteric
coatings prevent
release of medication before it reaches the small intestine. Most enteric
coatings work by
presenting a surface that is stable at acidic pH but breaks down rapidly at
higher pH.
Enteric coating of capsules filled with composition containing endoxifen or
endoxifen-lipid
complex can be done as methods known in the art.
[00121] The endoxifen -lipid complex of the present invention can be of
varying size
or can be of substantially uniform size. For example, the complex can have a
mean
diameter of about 1 mm or less, and more preferably are in the micron or sub-
micron range.
In some preferred embodiments, the complexes have an average diameter of about
5 pm or
less, such as 0.2 pm or less or 0.1 m or less.
[00122] The technology outlined in the present invention may also be used for
any
other water-insoluble drugs. The methods and compositions of the present
invention find
use in conjunction with the methods and compositions disclosed in
International Publication No. WO/2008/127358, filed October 10, 2007 and
International
Publication No. WO/2008/058156,filed November 6, 2007.
[00123] The compositions of the present invention can be employed to treat
breast
cancer and breast related diseases. For example, the compositions of the
present invention
may be administered to a patient diagnosed with benign breast disease. As used
herein, the
term" benign breast disease" refers to a constellation of non-malignant
aberrations in breast
tissue. The aberrations may be proliferative or non-proliferative in nature.
The exemplary
24
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
benign breast diseases treatable by the present inventive compositions include
adenosis,
cysts, duct ectasia, fibroadenoma, fibrosis, hyperplasia, metaplasia and other
fibrocystic
changes. Each of these diseases, referred as "changes" or "conditions" due to
their
prevalence, have well-defined histological and clinical characteristics.
[00124] "Adenosis" refers to generalized glandular disease of the breast. It
typically
involves an enlargement of breast lobules, which contain more glands than
usual. In
"sclerosing adenosis," or "fibrosing adenosis," the enlarged lobules are
distorted by scar-like
fibrous tissue.
[00125] "Cysts" are abnormal sacs filled with fluid or semi-solid material.
Cysts in
the breast arelined by breast epithelial cells, developing from lobular
structures. They begin
as excess fluid inside breast glands, but may grow to proportions that stretch
surrounding
breast tissue, causing pain. "Fibrocysts" are cystic lesions circumscribed by,
or situated
within, a conspicuous amount of fibrous connective tissue.
[00126] "Duct ectasia" refers to a dilation of mammary ducts by lipid and
cellular
debris. Rupture of the ducts induces infiltration by granulocytes and plasma
cells.
[00127] "Fibroadenoma" refers to benign tumors that are derived from glandular
epithelium and contain a conspicuous stroma of proliferating fibroblasts and
connective
tissue.
[00128] "Fibrosis" simply refers to a prominence of fibrous tissue in the
breast.
[00129] "Hyperplasia" refers to an overgrowth of cells, where several layers
of cells
line the basal membrane, without tumor formation. Hyperplasia increases the
bulk of
mammary tissue. In "epithelial hyperplasia," the cells lining breast ducts and
lobules are
involved, giving rise to the terms "ductal hyperplasia" and "lobular
hyperplasia." Based on
a histological determination, hyperplasia may be characterized as "usual" or
"atypical".
[00130] "Metaplasia" refers to a phenomenon in which a differentiated tissue
of one
type transforms into a differentiated tissue of another type. Metaplasia often
results from an
environmental change, and enables cells better to withstand the change.
[00131] The compositions of the present invention may be administered in any
dosage form and via any system that delivers the active compound endoxifen to
breast
estrogen receptors in vivo. In some embodiments, a composition of present
invention is
delivered by "percutaneous administration", e.g., delivering the drug from the
surface of
patient's skin, through the stratum corneum, epidermis, and dermis layers, and
into the
microcirculations. This is generally accomplished by diffusion down a
concentration
gradient. The diffusion may occur via intracellular penetration (through the
cells),
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
intercellular penetration (between the cells), transappendageal penetration
(through the hair
follicles, sweat, and sebaceous glands), or any combination of the above.
[00132] Percutaneous administration of the endoxifen composition of the
present
invention may be advantageous because this may reduce systemic drug exposure
and the
risks from non-specifically activating estrogen receptors throughout the body.
This is
because in topical application of endoxifen will absorb primarily into local
tissues. When
the composition of invention containing endoxifen will be percutaneously
applied to
breasts, high concentration will accumulate in the breast tissues presumably
due to many
estrogen receptors therein. The composition of endoxifen may be applied to any
skin
surface, preferably to one or both breasts. The daily doses to be administered
can initially
be estimated based upon the absorption coefficients of endoxifen, the breast
tissue
concentration that is desired, and the plasma concentration that should not be
exceeded.
The initial dose may be optimized in each patient, depending on individual
responses.
[00133] Percutaneous administration can be achieved in different ways, such as
(i) by
mixing the composition of endoxifen with suitable pharmaceutical carriers and,
optionally,
penetration enhancers to form ointments, emulsions, gel, lotion, creams or the
like, where
an amount of said preparation is applied onto a certain area of the skin, (ii)
by incorporating
the composition of endoxifen into patches or transdermal delivery systems
according to the
technology known in the art.
[00134] The effectiveness of percutaneous drug administration depends on many
factors, such as drug concentration, surface area of application, time and
duration of
application, skin temperature, skin hydration, previous irradiation,
physicochemical
properties of the drug, and partitioning of the drug between the formulation
and the skin. In
some embodiments, e.g., to enhance percutaneous effectiveness, the
compositions or
complexes comprise penetration enhancers that improve percutaneous absorption
by
reducing the resistance of stratum corneum by reversibly altering its
physicochemical
properties, changing hydration in the stratum corneum, acting as co-solvent,
or changing the
organization of lipids or proteins in the intracellular spaces. Such enhancers
include but are
not limited to organic solvents such as alcohol, acetone, dimethylsulfoxide
(DMSO),
polyethylene glycol, propoylene glycol, fatty acids and fatty alcohol and
their derivatives,
hydroxyl acids, pyrrolidones, urea, vegetable oils, essential oils, and
mixture thereof. In
addition to chemical enhancers, physical methods can increase percutaneous
absorption. For
example, occlusive bandages induce hydration of the skin. Other physical
methods include
iontophoresis and sonophoresis, which use electrical fields and high-frequency
ultrasound,
26
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
respectively, to enhance absorption of drugs that are poorly absorbed due to
their size and
ionic characteristics (12-13). Those who are in the pharmaceutical field can
easily
manipulate the various factors and methods to achieve right efficacious dosage
for
percutaneous delivery.
[00135] For percutaneous administration, the formulation or composition of the
invention containing endoxifen may be delivered in the form of ointment,
emulsion (lotion),
cream, gel, powder, oil or similar formulation. In some embodiments, the
formulation
comprises excipient additives, including but not limited to vegetable oils
such as soybean
oil, mustard oil, almond oil, olive oil, groundnut oil, peanut oil, peach
kernel oil, groundnut
oil, castor oil, canola oil, and the like, animal fats, DMSO, lanolin lipoids,
phosphatides,
hydrocarbons such as paraffin's, petroleum jelly, waxes, lecithin, detergent
emulsifying
agents, carotin, alcohols, glycerol, glycerol ether, glycerine, glycol, glycol
ethers,
polyethylene glycol, polypropylene glycol, non-volatile fatty alcohols, acids,
esters, volatile
alcoholic compounds, talc, urea, cellulose derivatives, coloring agents,
antioxidants and
preservatives.
[00136] In some embodiments the formulation or composition of the invention
containing endoxifen may be delivered as transdermal patch. The patch may
comprise (i) a
solution-impermeable backing foil, (ii) a layer like element having a cavity,
(iii) a
microporus or semipermeable membrane, (iv) a self-adhesive layer, and (v)
optionally a
removable backing film. The layer-like element having a cavity may be formed
by the
backing foil and the membrane. Alternatively, the patch may comprise(i) a
solution-
impermeable backing foil.(ii) an open-pored foam, a closed pore foam, a tissue
like layer or
a fibrous web-like layer as reservoir,(iii) a self adhesive layer, and(iv)
optionally a
removable backing film.
[00137] In some preferred embodiments, the composition of the invention
containing
endoxifen is formulated in hydro alcoholic gel and the amount of endoxifen may
vary from
0.001001 to 1.0 gram per 100grams of gel, most preferably in the range of 0.01-
0.20 grams
per 100 grams of gel.
[00138] In other embodiments, the composition of present invention comprises
one
or more fatty acid esters as a penetration enhancer. One of the highly
preferred examples of
a fatty acid ester penetration enhancer is isopropyl myristate. When isopropyl
myristate is
used in gel, the amount may range e.g., from 0.11 to 5.0 grams per 100 grams
of gel,
preferably from 0.5 to 2.Ograms per 100 grams of gel.
27
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
[00139] In another preferred embodiment the composition of invention
containing
endoxifen may also contain one or more nonaqueous vehicles, such as alcoholic
vehicles.
Examples of nonaqueous vehicles include ethyl acetate, ethanol, and
isopropanol,
preferably ethanol and isopropanol. These nonaqueous vehicles may be useful
for
dissolving both the active agent endoxifen and any other penetration enhancer
used. They
also preferably have a low boiling point, preferably less than 1000 C at
atmospheric
pressure, to permit rapid evaporation upon contact with skin. In particular,
ethanol may
effectively contribute to the percutaneous absorption of endoxifen by rapidly
evaporating
upon contact with skin. The amount of absolute nonaqueous vehicle in a gel
formulation
ranges from 35% to 99% by weight, preferably between 50% to 85% and more
preferably
between 60% to 75%.
[00140] In another preferred embodiment, the composition or formulation of the
invention comprises an aqueous vehicle that permits solubilization of
hydrophilic
molecules, and promotes moisturization of skin. An aqueous vehicle also can
regulate pH.
Aqueous vehicles include alkalinizing and basic buffer solutions, including
phosphate
buffer solutions, including phosphate buffer solutions (e.g., dibasic or
monobasic sodium
phosphate); citrate buffered solutions (e.g., sodium citrate or potassium
citrate) and purified
water. The amount of an aqueous vehicle preferably ranges between 0.1% to 65%
by
weight of the pharmaceutical composition, preferably between 15% to 50%, and
more
preferably between 25% to 40%.
[00141] In other embodiments, the composition of the invention comprises one
or
more gelling agents to increase the viscosity of the composition or
formulation or to
function as a solubilizing agent. It may constitute between 0.1 % to 20% by
weight of
formulation depending on the nature of gelling agent, preferably between 0.5%
to 10% and
more preferably between 0.5% to 5%. The gelling agents may be carbomers,
cellulose
derivatives, poloxamers and poloxamines. The preferred gelling agents are
chitosan,
dextran, pectins, natural gums and cellulose derivatives such as ethyl
cellulose,
hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl
cellulose (HPMC),
carboxymethyl cellulose (CMC) and the like. The most preferred gelling agent
is
hydroxypropyl cellulose.
[00142] The composition of invention may comprise a gelling agent as described
above, in particular a non-preneutralized acrylic polymer and also comprise a
neutralizing
agent. The ratio of neutralizing agent/gelling agent varies in between 10:1 to
0.1:1,
preferably between 7:1 to 0.5:1, and more preferably between 4:1 to 1:1 .A
neutralizing agent
28
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
in the presence of polymer should form salts that are soluble in the vehicle.
A neutralizing
agent also should permit optimum swelling of polymer chains during
neutralization of
charges and formation of polymer salts. The neutralizing agents include
ammonium
hydroxide, potassium hydroxide, sodium hydroxide, aminomethylpropanol,
trolamine, and
tromethamine. Those skilled in the art will select a neutralizing agent
according to the type
of geling agent used in the composition or formulation. However, no
neutralizing agent is
required when a cellulose derivative will be used as geling agents.
[00143] In some embodiments, the compositions of present invention are
employed
to treat other diseases, and the medication is selected from a lipophilic or a
compound made
lipophilic by derivatization of the group consisting of antiasthama,
antiarrhythmic,
antifungals, antihypertensive, anticancer, antibiotics, antidiabetics,
antihistamines,
antiparasitics, antivirals, cardiac glycosides, hormones, immunotherapies,
antihypotensives,
steroids, sedatives and analgesics, tranquilizers, vaccines, and cell surface
receptor blockers.
[00144] All references, including publications, patent applications, and
patent cited
herein, including those in the list below and otherwise cited in this
specification, are hereby
incorporated by reference to the same extent as if each reference was
individually and
specifically indicated to be incorporated by reference and were set forth in
the entirely
herein.
[00145] The use of terms "a" and "an" and "the" and similar referents in the
context
of describing the invention (especially in the context of the following
claims) are to be
construed to cover both the singular and the plural, unless otherwise
indicated herein or
clearly contradicted by context. The terms "comprising", "including",
"having", and
"containing" are to be construed as open-ended terms (i.e. meaning "including
but not
limited to") unless otherwise noted. The use of any and all examples, or
exemplary
language (e.g., "such as") provided herein, is intended merely to better
illuminate the
invention and does not pose a limitation on the scope of the invention unless
otherwise
claimed. No language in the specifications should be constructed as indicating
any non-
claimed element as essential to the practice of the invention.
[00146] Preferred embodiments of this invention are described, including the
best
mode known to the inventors for carrying out the invention. Variations of
those preferred
embodiments can become apparent to those of ordinary skilled artisans to
employ such
variations as appropriate, and the inventors intend for the inventions to be
practiced
otherwise than specifically described herein. Accordingly, this invention
includes all
modifications and equivalents of the subject matter recited in the claims
appended hereto as
29
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
permitted by applicable law. Moreover, any combination of the above-described
elements
in all possible variations thereof is encompassed by the invention unless
otherwise indicated
herein or otherwise clearly contradicted by context.
EXAMPLES
[00147] The following examples further illustrate the invention and are not to
be
construed as in any way as limiting its scope.
EXAMPLE 1
Synthesis of Compound 3
Br
Br
+ I H2SO4 O O O
Oo C-RT ""a
OH
1 2 3
[00148] 4-Bromophenol (1, 1 kg) and 3, 4-dihydro-2H-pyran (2, 1.5 L) was mixed
together in a round bottom flask and cooled to 0 C. Conc. Sulfuric acid (1 mL)
was added
drop wise while maintaining the temperature below room temperature. The
solution was
stirred at RT for 1 hr. The reaction solution was diluted with hexane and
washed with water
(1 L) followed by 5% sodium bicarbonate solution (1 L). The organic layer was
dried over
sodium sulfate, filtered and evaporated in vacuo at 50-55 C to give an oil
(1.55 Kg).
Hexane (300 mL) was added to the oil and triturated to give white solid 3. The
suspension
was cooled to 0 C and stirred for 30 min before it was filtered and washed
with cold hexane
(100 mL) and dried. Yield 1.32 Kg.
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
EXAMPLE 2
Synthesis of Compound 5
CI
Br CI I / \ O
\ ~ HI
+ I / \ O ::T:fI:X
O O
O
3 4 5
[00149] Magnesium turnings (115 g) were added to a 10_L 4-neck round bottom
flask containing anhydrous tetrahydrofuran (1 L). The mixture was heated to 55
C. Iodine
chips (approx. 5) were added in one lot followed by ethyl bromide (5 mL).
Compound 3
(1.1 kg) was dissolved in THE (2 L). 200 mL of this solution was added at once
to Mg-
THE suspension. The reaction was initiated after 30 mins and reflux started.
Remaining
solution of compound 3 was added drop wise maintaining the reflux temperature
over a
period of 1.5 h. The reaction mixture was further refluxed for 2 hr and the
cooled to RT.
(2-Chloroethoxyphenyl) phenyl butanone (4, 870 g) in THE (1.5 L) was added
drop wise
over a period of 1 h maintaining the temperature between 30-35 C. The
reaction mixture
was refluxed for 4h and cooled to RT. The reaction mixture was poured into ice
cold 50%
hydrochloric acid (3L). The organic layer was separated and the aqueous layer
was
extracted with THE (3 x 500 mL). The organic layers were combined, dried over
sodium
sulfate, filtered and concentrated to give 5 as oil which was carried over to
next step without
further purification. Yield - 1.57 kg.
25
31
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
EXAMPLE 3
Synthesis of Compound 6
CI
CI
OH I
HCI~ I / \ O
O O
OH
6
5 [00150] Compound (5, 1.57 kg) was dissolved in methanol (6 L) and cone.
hydrochloric acid (1.57 kg) was added. The solution was refluxed for 5 h.
Methanol was
removed in vacuo and dichloromethane (5 L) was added. The organic layer was
separated.
The aqueous layer was extracted with dichloromethane (2 x 500 mL). The organic
layers
were combined and washed with water (2 L), 5% aq. NaHCO3 (2 L), water (2L),
dried over
sodium sulfate. Charcoal was added and filtered. The solvent was removed under
vacuum
to give oil (1.38 kg). The oil was triturated with hexane (5 L) with vigorous
stirring to yield
6 as solid product which was filtered and dried. Yield 1.07 kg.
EXAMPLE 4
Synthesis of Compound I
CI
H3CHN
O Cj
O
CH3NH2
OH
OH
6
[00151] To a solution of compound 6 (50 g) in isopropanol (500 mL), monomethyl
amine (300 mL) was added and heated for 24 h maintaining the temperature
between 70-
32
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
75 C. The completion of reaction was monitored by TLC (toluene:triethylamine,
7:3). The
solvent was removed in vacuo. Water (500 mL) was added to the residue and
extracted
with diisopropyl ether (DIPE, 500 mL). The organic layer was separated and the
aqueous
layer was back extracted with DIPE (200 mL). The organic layers were combined
and
washed with water (500 mL), 5% aq. sodium bicarbonate (500 mL), dried over
sodium
sulfate and filtered. The solvent was removed in vacuo to give a gummy
residue. Ethyl
acetate (50 mL) was added and heated to dissolve the residue completely. The
solution was
cooled to RT and hexane (50 mL) was added and stirred for 12 h. The solid was
filtered
and washed with cold ethyl acetate-hexane (1:1, 10 mL) mixture. Product I was
dried
overnight under high vacuum. Yield 25 g.
EXAMPLE 5
Endoxifen Solution
[00152] Endoxifen solution (1 mg/mL) was prepared by solubilizing endoxifen
(10.3
mg) in 0.2% glacial acetic acid (10 mL). The pH (-5.75)of the solution was
adjusted with
IN sodium hydroxide (300 L).
EXAMPLE 6
Endoxifen Solution
[00153] Endoxifen solution (5 mg/mL) was prepared by solubilizing endoxifen
(100
mg) in 2% glacial acetic acid (8.6 mL). The solution was diluted with 5%
dextrose (10.97
mL). The pH (-5.56) of the solution was adjusted with 5N sodium hydroxide (430
L).
EXAMPLE 7
Endoxifen Complexes
[00154] A suspension of endoxifen, cholesteryl sulfate, and soy lecithin, is
produced
by mixing the components together in water and homogenizing using. e.g., a
high pressure
homogenizer. The resulting suspension can be filtered through 0.2 gm filter
and then mixed
with 7.5% sucrose solution and lyophilized in either vials or in bulk. The
particle size of
the resulting complexes is determined using standard procedures, e.g., using a
Nicomp
particle sizer 380.
33
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
EXAMPLE 8
Endoxifen Complexes
[00155] A suspension of endoxifen and soy lecithin is produced by mixing the
components together in water and homogenizing using, e.g., a high pressure
homogenizer..
The resulting suspension can be filtered through 0.2 gm filter and then mixed
with 7.5%
sucrose solution and lyophilized in eithervials or in bulk. The particle size
is determined
using standard procedures, e.g., using a Nicomp particle sizer 380.
EXAMPLE 9
Toxicity Testing
[00156] Endoxifen was formulated according to Example 6 and was tested for
toxicity in male Balb/c mice. A single test dose at 100 mg/kg or 50 mg/kg was
intravenously administered to mice. All the mice died at the 100 mg/kg dose
level whereas
all animals survived at the 50 mg/kg dose level with no significant loss of
body weight. The
mice also survived in the control group with a vehicle control that lacked
endoxifen. Repeat
dose toxicity study was conducted with a dose of 25 mg/kg administered
consecutively for 3
days with accumulated dose of 75 mg/kg. All the animals in this group
survived. The
results are reported in the table below as the number of mice surviving per
total.
Treatment Dose (mg/kg) Survival/Total
100 0/2
Single dose
50 1/4
Repeat dose 5 /4
EXAMPLE 10
Endoxifen exhibits anti-proliferative activity against different tumor cells
[00157] Endoxifen was tested for antiproliferation activity against various
cancer cell
lines from Non Small Lung Cancer, Breast Cancer, Prostate Cancer, Melanoma
Cancer,
Ovarian Cancer, CNS Cancer, Renal Cancer and Colon Cancers. The cells were
incubated
for multiple days (3 - 7) with Endoxifen (10 nM to 10 M) and the inhibition
of growth
were measured by SRB or MTT staining method. The results indicated significant
growth
inhibition of cells in the presence of Endoxifen ranging from 10 to 100%.
Endoxifen induce
34
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
growth inhibition or cell killing in different tumor cells indicates the
usefulness of
Endoxifen in the treatment of cancers in humans.
EXAMPLE 11
Endoxifen Inhibits Estradiol Dependent Breast Tumor Growth
[00158] It is known that Tamoxifen antagonizes estradiol-dependent breast
cancer
xenograft growth (15). Endoxifen base and endoxifen-citrate in oral dosage
form can be
similarly be tested for inhibition of estradiol dependent MCF-7 xenograft
growth. For the
animal experiments, female nude mice (Bom: NMRI-nu/nu) per xenograft
experiment, ages
4 to 6 weeks and weighing 20 to 24 g, are used according to standard
protocols. An
example of such a procedure is as follows:
[00159] MCF-7 xenografts are developed by passage of transplantable tumor from
a
parent tumor established in oophorectomized athymic nude mice treated with
estradiol (16-
17).
[00160] Randomly bred female athymic mice are bilaterally ovriectomized and
allowed a 2-week recovery period before the implantation of tumor material.
The s.c.
transplantation of the MCF-7 tumor fragments (size, 1 x 1 x 1 mm3) is done
under
anesthesia. The diameter of the tumors is measured regularly, e.g., once
weekly, using a
caliper-like mechanical instrument and the tumor volume (k) is calculated
according to the
empirical equation V= (length x width )/2. The median volumes of each group
are
normalized to the initial tumor volume resulting in the relative tumor volume.
In all the
experiments, tumor-bearing mice receive estradiol supplementation [estradiol
valeriate
(E2D), 0.5 mg/kg once/wk i.m.]. This supplementation leads to physiologic
levels of serum
E2 (25-984 pg/mL) that are comparable to the human situation (25-600 pg/mL
depending
on the follicular phase).
[00161] Substances: The following substances are used: E21), Tamoxifen and
endoxifen.
[00162] Treatment Modalities: All MCF-7 transplanted animals receive E2D (0.5
mg/kg) injections once a week. After 4 weeks, when hormone-supplemented tumors
have
grown to -0.7-0.8 cm in diameter (180-250 mm3), the mice are randomized into 4
treatment
groups of 5-10 mice each. The 5-10 mice are sacrificed as baseline controls
for E2D alone.
[00163] The treatment groups are: (i) E2D support (0.5mg/kg once/wk i.m); (ii)
E2D support (0.5mg/kg once/wk i.m) plus Tamoxifen (0.5mg-2mg)/mouse per day, 5
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
days/week by gavage ; (iii) E2D support (0.5mg/kg once/wk i.m) plus endoxifen
(0.5mg-
2mg)/mouse per day, 5 days /week by gavage; (iv) withdrawal of E2D support.
[00164] Suppression of tumor growth in this breast cancer tumor model is
indicative
of therapeutic effect in the treatment of breast cancer in humans (15).
EXAMPLE 12
Endoxifen Minimizes Uterotrophic Effect of Estrogen
[00165] It is known that Tamoxifen is a non-steroidal agent with potent anti-
estrogenic effect in animal and in vitro models. This pharmacologic property
is related to
the drug's ability to compete with estrogen for estrogen receptors in breast
tissues, and to
inhibit the stimulatory effect of estrogen on the uterus, vagina and ovaries
(18).
[00166] Endoxifen (0.1 mg-2 mg) is administered orally once daily for 28 days
to
determine the reduction in utertrophic effect of estradiol; Female BALB/c mice
approximately 50 days old and weighing 19-20 g are obtained (e.g., from
Charles-River,
Inc.) and housed four to five per cage at a temperature (23+_ 1 C) and light
(12 h light/
day). The atrophic changes are observed in the mice. There will be three
groups such as
vehicle control, Tamoxifen and endoxifen. The animals (5-10 mice) are randomly
assigned
to each group. Daily treatments of intact mice with a dose (e.g., 0.1 mg-2mg)
by gavage of
Tamoxifen or endoxifen are expected to lead to progressive inhibition of
uterine and vaginal
weight.
[00167] Such results will show that endoxifen has better minimizing
uterotrophic
effect of estrogen than Tamoxifen, and that endoxifen finds use as an
effective anti-
estrogen. Endoxifen blocking of uterine weight gain stimulated by estrogen can
also be
demonstrated in immature rats. Endoxifen preparations showing the effects
described
above find use in the treatment of breast cancer as well as other estrogen-
sensitive
conditions, such as endometriosis, leiomyomata, and benign breast disease, as
well as other
estrogen-responsive conditions in men and women.
EXAMPLE 13
Endoxifen-Caused Decrease of Ki-67 Antigen Expression in Proliferating Breast
Cancer Cells
[00168] Ki-67 is a nuclear non-histone protein. This antigen is absent in
quiescent
cells and is expressed in proliferating cells and is used as a biomarker (19-
20). Endoxifen
36
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
base or endoxifen-citrate in oral or injectable form are given to xenograft
breast cancer
tumor models (e.g., as described above), as well as to breast cancer patients.
Immunochemical determination of Ki-67 is done in tumor cells from breast
cancer tissues
from patients, as well as from mice bearing tumors as described in Example 11.
The MIB-1
or similar antibody available from commercial sources such as DAKO,
Carpenteria, CA is
used for immunochemical localization of antigen. Decrease in Ki-67 antigen
expression in
animals and/or breast cancer patients demonstrate the applicability of
endoxifen in treating
breast cancers.
EXAMPLE 14
Endoxifen reduces IGF-1 levels in Breast Cancer
[00169] It is known for humans that Tamoxifen reduces the levels of
circulating
insulin-like growth factor I (IGF- 1). IGF-1 has been used as a surrogate
biomarker and
predicts the effectiveness of Tamoxifen in treatments of breast cancer
patients (21). To test
the effects of endoxifen preparations of the present invention, endoxifen base
or endoxifen-
citrate are given orally or injected to experimental animals bearing breast
cancer tumors.
The concentration of IGF-1 levels in control and xenografted breast tumor is
monitored by
established assays (e.g., ELISA Kit from Diagnostics Systems Laboratories,
London, UK or
DAKO, Carpenteria, CA). Endoxifen is administered by gavages at 0.5mg-2mg per
mouse
per day, 5 days/week. Decrease of IGF-1 levels and tumor growth reduction
indicates the
usefulness of IGF-1 as a surrogate marker for breast cancer.
EXAMPLE 15
Endoxifen Prevents Development of Bicalutamide -induced
Gynecomastia and Breast Pain
[00170] Bicalutamide (CasodexR) is used for treating prostate cancer in men.
There
is growing evidence that IGF-1 may be involved in prostate cancer promotion
and
progression. It is also known that anti-estrogen agents such as Tamoxifen
decrease IGF-1
levels and prevent biculatamide -induced gynecomastia in prostate cancer
patients (22).
Since, endoxifen is an active metabolite of the Tamoxifen anti-estrogen, the
silastic slow-
release capsules containing endoxifen for implant or oral doses of endoxifen
(lmg-
l Omg/day) with biculatamide are expected to prevent development of
biclutamide -induced
gynecomastia and breast pain.
37
CA 02669913 2011-09-15
REFERENCES
1. Furr BJ, Jordan VC. The pharmacology and clinical uses of Tamoxifen.
Pharmacol
Ther 1984; 25:127-205.
2. Osborne. Tamoxifen in the treatment of breast cancer. N Engl J Med 1998;
339:1609-18.
3. Fisher B, Costantino JP, Wickerham DL, Redmond CK, kavanah M, Cronin WM, et
al. Tamoxifen for prevention of breast cancer: Report of the National Surgical
Adjuvant and Bowel Project P-1 Study. J. Natl Cancer Insts 1998; 90:1371-88.
4. Stearns V, Ullmer L, Lopez JF, Smith Y, Issacs C, Hays D. Hot flushes.
Lancet
2002; 360:1851-61.
5. Otton SV, Ball SE, Cheung SW, Inaba T, Rudolph RL, Sellers EM. Venlafaxine
oxidation in vitro is catalyzed by CYP2D6. Br J Clin Pharmacol 1996; 41:149-
56.
6. Steams V, Johnson MD, Rae JM, Morocho A, Novielli A, Bhargava P,
Hayes DF, Desta Z, Flockhart DA. Active Tamoxifen metabolite concentrations
after co-administration of Tamoxifen and the selective serotonin reuptake
inhibitor
paroxetine. J Natl Cancer Inst 2003; 95:1758-1764.
7. YC, Desta Z, Flockhart DA and Skaar TC. Endoxifen (4-hydroxy-N-desmethyl-
Tamoxifen) has anti-estrogenic effects in breast cancer cells with potency
similar to
4-hydroxy-Tamoxifen. Journal of Cancer Chemotherapy and Pharmacology 2005:
55: 471-478.
38
CA 02669913 2011-09-15
8= Young Chai Lim, Lang Li, Zeruesenay Desta, Qianqian Zhao, James M. Rae,
David
A. Flockhart, and Todd C. Skaar. Endoxifen, a Secondary Metabolite of
Tamoxifen,
and 4-OH-Tamoxifen Induce Similar Changes in Global Gene Expression Patterns
in MCF-7 Breast Cancer Cells; JPET, 2006, 318:503-512.
9. Matthew P. Goetz, James M. Rae, Vera J. et al. Pharmacogenetics of
Tamoxifen
Biotransformation Is Associated With Clinical Outcomes of Efficacy and Hot
Flashes. JCO Dec 20 2005: 9312-9318.
10. Satyavati GV, Economic and Medical Plant Research, 5, 47(199 1).
11. Indian Pharmacopea, 1988.
12. Remington: The Science and Practice of Pharmacy (Lippincott Williams &
Wilkins
(2000), pages 836-58.
13. Percutaneous Absorption: Drugs Cosmetics Mechanisms Methodology edited by
Bronaugh and Maibach, Marcel Becker Publisher (1999).
39
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
15. Stephen R.D. Johnston, Irene M. Boeddinghaus, Sharon Riddler, Ben P.
Haynes, Ian
R. Hardcastle, Martin Rowlands, Rachel Grimshaw, Michael Jarman, and Mitch
Dowsett. Iodoxifen Antagonizes Estradiol-dependent MCF-7 Breast Cancer
Xenograft Growth through Sustained Induction of Apoptosis, Cancer Research,
59,
3646-3651 (1999).
16. Iino, Y, Wolf, DM, Langan-Fahey SM, Johnson, DA, Ricchio, M, Thompson, ME,
and Jordan VC. Reversible Control of Oestradiol-stimulated Growth of MCF-7
Tumours in the Athymic Mouse. Br. J. cancer, 64: 1019-1024 (1991)
17. Jann N Sarkaria, David FC Gibson, V C Jordan, John F Fowler, Mary J.
Lindstrom,
and R. Timothy Mulacahy. Tamoxifen-induced increase in the Doubling time of
MCF-7 Xenografts as Determined by Bromodeoxyuridine Labeling and Flow
Cytometry. Cancer Research 53, 4413-4417 (1993).
18. Nanjoo Suh, Andrew L. Glasebrook, Alan D. palkowitz, Henry U. Bryant,
Lorris L.
Burris, james J. Starling, Homer L. Pearce, Charlotte Williams, Christopher
Peer,
Yongping Wang and Michael B. Sporn. Arzoxifene, a New Selective Estrogen
receptor Modulator For Chemo prevention of Experimental Breast Cancer. Cancer
Reseearch 61, 8412-8415 (2001).
19. Assersohn L, Salter J, Powles TJ, et al: Studies of the potential utility
of Ki-67 as a
predictive molecular marker of clinical response in primary breast cancer.
Breast
Cancer Res Treat 82:113-123, ( 2003).
20. Kenny FS, Willsher PC, Gee JM, et al: Change in expression of ER, bcl-2
and MIB-
1 on primary Tamoxifen and relation to response in ER positive breast cancer.
Breast Cancer Res Treat 65: 135-144, (2001).
21. Nahta R, Hartobagyi GN and Esteva FJ. Growth factor Receptors in Breast
Cancer:
Potential for Therapeutic Intervention, Oncologist 8: 5-17, (2003).
22. Saltstein D, Sieber P, Morris T, Gallo J. Prevention and Management of
Biclutamide-induced gynecomastia and Breast Pain: Randomized Endocrinologic
CA 02669913 2009-05-15
WO 2008/070463 PCT/US2007/085443
and Clinical Studies with Tamoxifen and Anastrozole. Prostate cancer and
Prostatic
Diseases 8, 75-83 (2005).
41
